The role of direct heparan sulfate interactions and Ca2+ complexation in Hedgehog gradient formation in vivo

发育生物学中的一个主要问题是细胞如何协调它们与邻居的行为。Hedgehog（Hh）形态发生蛋白是果蝇发育过程中细胞通讯的中心模型，它从其来源扩散到远处的应答细胞和组织中，指导其生长和模式形成。未受损的Hh转运和梯度形成需要细胞外表达的线性和高度带负电荷的糖链称为硫酸乙酰肝素（HS）。然而，HS结合并固定大多数其他细胞外可溶性蛋白，这就提出了一个问题，即与HS的相互作用如何促进Hh的传播，而不是减缓Hh的传播。在最后一个资助期间，我们解决了这个矛盾，表明直接静电Hh与细胞表面HS的相互作用启动和引导Hh运输以类似的方式静电约束DNA结合蛋白在细胞核中的运动。DNA结合蛋白最好沿双螺旋的轴沿着移动，两个DNA结合位点允许糖-磷酸骨架之间的直接转移，以提高搜索速度。使用在硅片，先进的体外和体内技术的组合，我们表明，HHS也直接开关之间的许多糖硫酸酯链在梯度场通过两个网站的密集排列的正电荷，和正电荷减少在一个网站严重损害Hh开关在体外和其运输到体内的遥远的目标。因此，我们发现，HS链之间直接切换的能力代表了一个以前未知的决定因素的时间编码的形态发生运动与其他HS结合蛋白的重要影响。在这个建议中，我们的目标是扰乱的组成和位置的带电氨基酸残基在无序的N-尾的Hh，使用我们建立在体外和体内的协议。我们还将研究动态HS的表达和硫酸化在发展中的组织，确定可访问的“微空间”的形态。我们预计，HS硫酸化增加的HS微空间在其周围组织上积累Hh，而表达具有较低硫酸化的HS的相邻微空间将不会进入。我们将可视化动态HS表达和硫酸化的变化，在发展中的翅膀和眼睛盘微空间，通过使用单链可变片段（scFv）抗体针对定义的HS结构。我们的第三个目标是继续表征的飞线不足的Ca 2 +-协调Hh氨基酸。这些额度是在上一个供资期间使用我们既定的协议生成的。他们的表型显示，Hh钙离子协调是非必需的苍蝇发展，但特别是干细胞增殖和苍蝇配子发生所需的。因此，在下一个资助期内，我们的目标是进一步表征Hh生物学的这一重要但知之甚少的方面。

A major question in developmental biology is how cells coordinate their behaviors with that of their neighbors. As a central model for cellular communication during development, Hedgehog (Hh) morphogens spread away from their source to direct growth and pattern formation in distant responding cells and tissues in Drosophila. Unimpaired Hh transport and gradient formation require extracellular expression of linear and highly negatively charged sugar chains called heparan sulfate (HS). However, HS binds and immobilizes most other extracellular soluble proteins, raising the question of how interactions with HS contribute to Hh spread instead of slowing it down. In the last funding period, we resolved this paradox by showing that direct electrostatic Hh interactions with cell-surface HS initiate and guide Hh transport in a similar way to electrostatically constrained DNA-binding protein movement in the nucleus. DNA-binding proteins preferably move along the axis of the double helix, and two DNA binding sites allow for direct transfer between sugar-phosphate backbones to increase search speed. Using a combination of in silico, advanced in vitro and in vivo techniques, we showed that Hhs do also switch directly between the many sugar-sulfate chains in the gradient field via two sites of densely arranged positive charge, and that positive charge reduction in one site severely impairs Hh switching in vitro and its transport to distant targets in vivo. Thus, we revealed that the ability to directly switch between HS chains represents a previously unknown determinant of temporally encoded morphogen movement with important implications for other HS-binding proteins. In this proposal, we aim to perturb the composition and position of charged amino acid residues in the disordered N-tail of Hh, using our established in vitro and in vivo protocols. We will also investigate dynamic HS expression and sulfation in developing tissues that determines accessible “microspaces” for morphogens. We expect that HS microspaces of increased HS sulfation over their surrounding tissue accumulate Hh, whereas neighboring microspaces expressing HS with lower sulfation will not be entered. We will visualize dynamic HS expression and sulfation changes in developing wing and eye disc microspaces by using single chain variable fragment (scFv) antibodies directed against defined HS structures. Our third aim is the continued characterization of fly lines made deficient in Ca2+-coordinating Hh amino acids. These lines were generated in the last funding period using our established protocol. Their phenotypes revealed that Hh Ca2+-coordination is non-essential for fly development, but is specifically required for stem cell proliferation and fly gametogenesis. In the next funding period, we therefore aim to further characterize this important yet poorly understood aspect of Hh biology.